Selective reduction of proteins

ABSTRACT

The present invention provides a method for making uncapped cysteine protein preparations, including uncapped engineered cysteine antibody preparations. The methods include, inter alia, contacting a reducing agent with engineered cysteine antibody molecules, each of the antibody molecules having at least one capped engineered cysteine residue and at least one interchain disulfide bond and reacting the reducing agent with the antibody molecules under conditions sufficient to uncap engineered cysteine residues and form cap byproducts. The method also includes removing the bap byproduct during the reduction reaction. Substantially all of the interchain disulfide bonds present in the antibody molecules prior to reduction are retained following reduction. Antibody conjugates and methods for preparing antibody conjugates using uncapped antibody preparations are also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/580,290, filed on Sep. 24, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/117,254 filed Aug. 8, 2016 (now U.S. Pat. No.10,464,997) which is a U.S. 371 National Stage Application ofinternational Application No. PCT/US2015/015369 filed Feb. 11, 2015,which application claims priority to U.S. Application Ser. No.61/938,378 filed Feb. 11, 2014, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Monoclonal antibodies in which selected amino acids have been mutated tocysteine (i.e., engineered cysteine mAbs, or ecmAbs) are particularlysuitable for use in conjugates (e.g., antibody drug conjugates (ADCs))because the conjugates derived from them can have favorable propertiesincluding homogeneity, favorable pharmacokinetics, stability, andsolubility. The cysteine mutations are placed in locations in the aminoacid sequence of the antibody which generally do not form inter- orintra-chain disulfide bonds, and expression machinery inside the cellproducing the mutant mAb treats the cysteine residues as unpairedcysteines. Consequently, the engineered cysteines are generallyexpressed in the form of mixed disulfides with non-encoded cysteinemolecules (i.e., the engineered cysteines are “capped” with cappingagents, e.g., cysteine, cysteinyl glycine, or glutathione).

Hence, in order to prepare conjugates (e.g., ADC) from ecmAbs, it isgenerally necessary to subject the ecmAb to reducing conditions toconvert the engineered cysteine from a mixed disulfide to a free thiol,and typically this uncapping or “activation” is accompanied by reductionof the ecmAb inter-chain disulfides. Although inter-chain disulfides canbe re-formed by mild oxidation, such re-oxidation steps add to thecomplexity and expense of ADC preparation. Selective reduction methodshave been elusive, as it has proven difficult to reduce the engineeredcysteines without simultaneously reducing the inter-chain disulfides.Consequently, selective reduction methods for uncapping of engineeredcysteine residues are needed. The present invention addresses this andother needs.

BRIEF SUMMARY OF THE INVENTION

The invention provides, inter alia, a method for selectively reducingengineered cysteine antibodies. The method includes contacting areducing agent with engineered cysteine antibody molecules, each of theantibody molecules having at least one capped engineered cysteineresidue and at least one inter-chain disulfide bond, and reacting thereducing agent with the antibody molecules under conditions sufficientto uncap engineered cysteine residues and form cap byproducts. Capbyproduct is removed during the reduction reaction. The methods resultin the formation of an uncapped engineered cysteine antibodypreparation. The uncapped engineered cysteine antibody preparation cancontain both capped and uncapped engineered cysteine antibody.Substantially all of the inter-chain disulfide bonds present in theantibody molecules prior to the reduction reaction are retained in theuncapped engineered cysteine antibody preparation. The method canfurther comprise the step of supplementing the reduction reaction withadditional reducing agent while removing cap byproduct. Removing the capbyproduct from the reaction mixture during the reduction reaction canbe, for example, via dialysis or diafiltration. In preferredembodiments, the concentration of the cap byproduct is maintained in thereduction reaction mixture below the concentration at which re-cappingprevents further activation of engineered cysteine residues

In some aspects, the methods provide an uncapped engineered cysteineantibody preparation. Typically, at least about 60% of the engineeredcysteine residues present in the antibody preparation are uncappedengineered cysteine residues. In some aspects, at least about 70%, atleast about 75% or at least about 80% of the engineered cysteineresidues present in the antibody preparation are uncapped engineeredcysteine residues. In some aspects, using the present methods, at least85% of the inter-chain disulfide bonds present in the antibody moleculesprior to the reduction reaction are retained in the uncapped engineeredcysteine antibody preparation. In some aspects, the reducing agent andreducing conditions are selected such that no more than about 20%, nomore than about 15%, no more than about 10% or no more than about 5% ofthe inter-chain disulfide bonds present in the antibody molecules areconverted during the reduction reaction to a pair of free thiols.

In related aspects, the invention provides antibody conjugates,including antibody drug conjugates, and methods for preparing antibodyconjugates using uncapped antibody. Residual reducing agent can beremoved from the antibody preparation prior to preparing antibody drugconjugates.

The invention also provides a method for selectively reducingnon-antibody proteins with unpaired cysteine residues. The methodincludes contacting a reducing agent with protein molecules, each of theprotein molecules having at least one capped cysteine residue and atleast one inter-chain disulfide bond, and reacting the reducing agentwith the protein molecules under conditions sufficient to uncap cysteineresidues and form cap byproducts. Cap byproduct is removed during thereduction reaction. The methods result in the formation of an uncappedcysteine protein preparation. The uncapped cysteine protein preparationcan contain both capped and uncapped cysteine protein molecules.Substantially all of the inter-chain disulfide bonds present in theprotein molecules prior to the reduction reaction are retained in theuncapped cysteine protein preparation. The method can further comprisethe step of supplementing the reduction reaction with additionalreducing agent while removing cap byproduct. Removing the cap byproductfrom the reaction mixture during the reduction reaction can be, forexample, via dialysis or diafiltration. In preferred embodiments, theconcentration of the cap byproduct is maintained in the reductionreaction mixture below the concentration at which re-capping preventsfurther activation of cysteine residues. The methods provide an uncappedcysteine protein preparation. Typically, at least about 60% of thecapped cysteine residues are uncapped (or, in other words, activated)using said methods. In some aspects, at least about 70%, at least about75% or at least about 80% of the capped cysteine residues are uncappedusing said methods. In some aspects, using said methods, at least 85% ofthe inter-chain disulfide bonds present in the protein molecules priorto the reduction reaction are retained in the uncapped engineeredcysteine antibody preparation. In some aspects, the reducing agent andreducing conditions are selected such that no more than about 20%, nomore than about 15%, no more than about 10% or no more than about 5% ofthe inter-chain disulfide bonds present in the protein molecules areconverted during the reduction reaction to a pair of free thiols.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a rPLRP column analysis of samples generatedby treating an ecmAb (99 μMin solution) with three different monothiolreducing agents (cysteine, N-acetyl cysteine (NAC), and cysteamine)under identical conditions at low concentration (2 mM). The Y axisrepresents the % activation of total engineered cysteines available.None of the reducing agents is able to approach 100% activation of theavailable engineered cysteine residues. There was no evidence ofreduction of inter-chain disulfide bonds in these experiments.

FIG. 2 shows the results of a rPLRP column analysis of heavy chain froma S239C ecmAb mcMMAF ADC. The S239C ecmAb was selectively reduced, bythe methods of the invention, and conjugated to the mcMMAF drug-linker.The reducing agent was cysteine at a concentration of 0.8 mM. Theactivation of the engineered cysteine residues proceeded essentially tocompletion. Bars marked ¾HO indicate heavy chain with no drug-linkerconjugated. Bars marked ¾HI indicate heavy chain with I drug-linkerconjugated and bars marked % H2 indicate heavy chain with 2 drug-linkersconjugated.

FIG. 3 shows the results of a rPLRP column analysis of heavy chain froma S239C ecmAb mcMMAF ADC. The S239C ecmAb was selectively reduced, bythe methods of the invention, and conjugated to the mcMMAF drug-linker.The reducing agent was cysteine at a concentration of 0.55 mM. Only asmall percentage of the engineered cysteine residues remained capped(¾HO), while the percentage of uncapped engineered cysteine residuesapproached I 00% (¾HI), and the amount of non-specific reduction (% H2)remained low.

FIG. 4 shows that activation of engineered cysteines was achievedselectively with S239C ecmAbs irrespective of the identity of the heavyand light chain variable regions. mAb I is the humanized 2H12 ecMAb andmAb2 is the humanized hlF6 ecMab (activation: ¾HO; selectivity: % H2).The rate of uncapping was similar for the two mAbs. Selective activationof engineered cysteines is demonstrated for mAb I at 3 differentmutation sites, 5239, K326 and A327. The results indicate thatengineered cysteine activation can be accomplished selectively at avariety of sites. These experiments were performed with −0.9 mMcysteine.

FIG. 5 shows the activation of engineered cysteines for the S239C mutantof mAb1. The mAb concentration in the reaction mixture was approximatelythe same as for the reactions in FIG. 4 , but the cysteine concentrationwas lower, approximately 0.5 mM throughout the time-course. Thisexperiment showed that maximal selectivity is obtained at a low cysteineconcentration, and that given sufficient time, complete activation canbe achieved with very good selectivity. The average drug load of theconjugate prepared from the final time point in this experiment was1.93, which is close to the nominal value of 2.0, and within the rangeof values obtained by the conventional method of non-selective reductionfollowed by re-oxidation.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides, inter alia, methods for selectivelyremoving sulfide caps from engineered cysteine residues in antibodies,including monoclonal antibodies.

Although some selective removal is observed after prolonged exposure ofantibodies to small molecule thiols at low concentrations, complete capremoval does not occur in a static reaction mixture. Advantageously,nearly complete activation of engineered cysteines can be achieved byutilizing the methods of the invention in which the reducing agentconcentration is maintained while reduction byproducts are removed. Inembodiments where it is not desired to activate substantially all of theengineered cysteines, the present methods can be used to control thelevel of activation. Surprisingly, the uncapped antibodies are obtainedwith interchain disulfide bonds intact, preserving antibody structureand eliminating the need for a reoxidation step prior to conjugation ofthe antibodies with drug or other functional agents. The methods of theinvention provide, inter alia, a significant simplification of currentmanufacturing practice for preparation of antibody conjugates, includingantibody drug conjugates.

II. Definitions

As used herein, the terms “antibody” broadly refers to intact monoclonalantibodies, polyclonal antibodies, monospecific antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that exhibit the desired biological activity (i.e., specificbinding to a target antigen) and that have at least one nativeinter-chain disulfide bond. Exemplary fragments include, for example,Fabs, minibodies and the like. An intact antibody is typically composedof four polypeptide chains (two heavy chains and two light chains), eachpolypeptide having primarily two regions: a variable region and aconstant region. The variable region specifically binds to and interactswith a target antigen. The variable region includes complementaritydetermining regions (CDRs) that recognize and bind to a specific bindingsite on a particular antigen. The constant region may be recognized byand interact with the immune system (see, e.g., Janeway et al., 2001,Immuno. Biology, 5th Ed., Garland Publishing, New York). The fourpolypeptide chains are covalently linked to each other via inter-chaindisulfide bonds. An antibody can be of any type (e.g., IgG, IgE, IgM,IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass. The antibody can be derived from any suitable species. In someembodiments, the antibody is of human or murine origin. A monoclonalantibody can be, for example, human, humanized, or chimeric. Dependingon the context, the term “antibody” can refer to a singular antibodymolecule or a collection of antibody molecules, such as in an antibodysolution.

As used herein, the term “inter-chain disulfide bond” refers to acovalent bond between two cysteine residues on adjacent polypeptidechains in an antibody. The disulfide bond has the formula R¹—S—S—R²,wherein the sulfur atoms are present in the cysteine sidechains and R¹and R² represent the remainder of the cysteine residues and thepolypeptide chains in which they reside. An inter-chain disulfide bondis generally present between a heavy chain and a light chain in anantibody, or between the two heavy chains.

As used herein, the term “engineered cysteine residue” refers to acysteine residue that is introduced into the peptide sequence of aprotein (e.g., antibody). A monoclonal antibody having an engineeredcysteine residue can be referred to as an “ecmAb.” The engineeredcysteine residue is generally not present in the native (i.e.,naturally-occurring) peptide sequence of the protein. The engineeredcysteine residue can take the place of the amino acid that naturallyoccurs at a given position in the peptide sequence, and can beintroduced into the peptide sequence via recombinant techniques such assite-directed mutagenesis. The engineered cysteine residue can be cappedor uncapped.

As used herein, the term “uncapped cysteine residue” refers to acysteine residue wherein the a-sidechain contains a free thiol moietyhaving the formula R¹—SH. R¹ represents the non-thiol portion of thecysteine residue. The uncapped cysteine residue can be an uncappedengineered cysteine residue.

As used herein, the term “capped cysteine residue” refers to a cysteineresidue wherein the a-sidechain contains a disulfide moiety having theformula R¹—S—S—R³. R¹ represents the non-thiol portion of the cysteineresidue, and R³ represents the non-thiol portion of a capping moietyhaving a molecular weight less than or equal to about 500 Da. The capcan be, for example, cysteine, cysteinyl glycine, or glutathione (withR³ representing the non-thiol portion of free cysteine, cysteinylglycine, or the non-thiol portion of glutathione, respectively) or anyother available monothiol. The capped cysteine residue can be a cappedengineered cysteine residue.

As used herein, the term “reduction reaction” refers to a reactionwherein a capped cysteine residue (e.g., capped engineered cysteineresidue) having the formula R¹—S—S—R³ is reduced and forms a thiolmoiety having a structure R¹—SH. R¹ and R³ are defined as in the abovedescriptions.

As used herein, the term “reducing agent” refers to a compound that canreduce disulfide bonds. Reducing agents include, but are not limited to,thiols such as cysteine, cystamine, and P-mercaptoethanol.

As used herein, the term “oxidizing agent” refers to a compound thatcauses the conversion of a pair of free thiols to a disulfide bond.Examples of oxidizing agents include, but are not limited to,5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dehydroascorbic acid (DHAA),and copper sulfate (CuSO4). “A re-oxidation step” is an affirmative stepthat is taken to cause the conversion of a pair of free thiols to adisulfide bond. Affirmative steps include introduction of an exogenousoxidizing agent and/or an intentional hold period to allow forautoxidation.

As used herein, “removing” a substance, such as a cap byproduct, from amixture containing an antibody and the substance refers to removing anyportion of the substance, including the entirety of the substance, fromthe mixture. Removing the substance can also include transferring theantibody from a first mixture containing the substance to a secondmixture not containing the substance. Removing a substance from amixture can include steps such as dialysis, diafiltration,chromatography, and the like.

As used herein, the terms “antibody-drug conjugate” and “ADC” refer toan antibody conjugated to a therapeutic agent, (i.e., a drug) optionallyvia a linker.

As used herein, the term “drug-linker compound” or “drug-linker” refersto a molecule having a drug moiety and a linker attached thereto,wherein the linker contains a reactive moiety suitable for attachment toan amino acid residue (such as a cysteine residue) in an antibody.

III. Description of the Embodiments

The present invention provides, inter alia, a method for selectivelyreducing engineered cysteine antibodies thereby forming an uncappedantibody preparation. The method includes contacting a reducing agentwith engineered cysteine antibody molecules, each of the antibodymolecules having at least one capped engineered cysteine residue and atleast one inter-chain disulfide bond and reacting the reducing agentwith the antibody molecules under conditions sufficient to uncapengineered cysteine residues and form cap byproducts. During thereduction reaction, the cap byproduct is removed, thereby preventingre-capping of the newly formed thiols. Reducing agent, eliminated alongwith the cap byproduct, is replaced to drive the reduction reactionforward. The reducing agent and conditions are selected such that theengineered cysteine residues are selectively reduced (i.e., selectivelyactivated). As a result, a separate re-oxidation step is not required inorder to reform inter-chain disulfide bonds following the reductionstep. Substantially all of the interchain disulfide bonds present in theantibody molecules having the capped engineered cysteine residues areretained in the uncapped antibody preparation. By use of the term“retain” it is not meant that the interchain disulfide bonds necessarilyremain intact during the reduction reaction. Bonds may be broken duringthe reduction but they reform prior to conversion into a pair of freethiols. Bond reformation is not dependent on a separate re-oxidiationstep but occurs during the reduction reaction as shown in Scheme 2.

Scheme I shows a schematic representation of cysteine residue uncappingaccording to the methods of the invention, with a monoclonal antibody asthe exemplary protein. As shown in reaction (i), reaction of a reducingagent (1) with an antibody having a capped engineered cysteine residue(2) results in an antibody having an uncapped engineered cysteineresidue (3) and a cap byproduct (4). Reaction (i) can proceed in bothdirections, and reaction of the uncapped engineered cysteine residuewith the cap byproduct can result in recapping of the engineeredcysteine residue. The result of the back reaction is a composition inwhich only some of the antibodies in the composition have an uncappedengineered cysteine residue. According to the methods of the invention,the reaction mixture is supplemented with additional reducing agent asshown in reaction (ii) and cap byproduct is removed from the reactionmixture. Reducing agent supplementation and cap byproduct removal candrive the reaction forward and prevent the back reaction, promoting theformation of the desired antibody having the uncapped engineeredcysteine residue (3).

Under normal (i.e. “static solution”) conditions, the re-cappingreaction limits the extent of activation of engineered cysteines. Thegraph in Figure I shows the results of treating an ecmAb with threedifferent monothiols under identical conditions. The Y axis representsthe % activation of total engineered cysteines available. The graphshows that none of the reducing agents is able to approach I 00%activation of the available engineered cysteine residues, and thislimitation is because the disulfide byproduct (4, Scheme I) re-caps thegenerated thiol, as described above.

Reducing Agents

Any suitable reducing agent can be used in the methods of the invention.Examples of reducing agents include, but are not limited to, monothiolreducing agents such as cysteine, N-acetyl cysteine, cysteamine,P-mercaptoethanol, 2-mercaptoethanesulfonic acid sodium salt, and thelike. Mild reducing agents such as cysteine and the like areparticularly suitable for removing the cap from a capped cysteineresidue (e.g., capped engineered cysteine residue) without reducinginterchain disulfide bonds. Stronger reducing agents, such asdithiothreitol (DTT), dithioerythritol (DTE), andbis(2-mercaptoethyl)sulfone), can reduce interchain disulfide bonds tooquickly in many cases. In particular, reducing agents such as TCEP andDTT, for which the mixed disulfide of intermediate 6 of Scheme 2 eitherdoes not form, or forms only transiently, are unlikely to selectivelyuncap capped cysteines. In some embodiments, the reducing agent isselected from cysteine, cysteamine, P-mercaptoethanol,2-mercaptoethanesulfonic acid sodium salt, and mixtures thereof.

Reaction Conditions

While many monothiols can be used for selective activation, the graph inFIG. 1 shows that different thiols activate engineered cysteines atdifferent rates, and to different maximal levels. Hence, identifyingsuitable conditions for activation of engineered cysteine residuesinvolves identifying a suitable reducing agent and identifying asuitable concentration of the reducing agent.

Reaction conditions will be determined, in part, by the location of theengineered cysteine residue, the identity of the cap, or residues in aparticular antibody. Solvent-exposed engineered cysteine residues, forexample, can be uncapped more readily than buried or partially-buriedengineered cysteine residues.

Conditions favoring selective activation of unpaired cysteine residuesin proteins can be understood based on the reactions in Scheme 2. If areducing reagent reacts with an engineered cysteine residue—as inreaction (a}—very much faster than with an inter-chain disulfide bond asin reaction (b), then selective activation may be relativelystraightforward. This condition occurs only, if ever, when theengineered cysteine is highly exposed to solvent on the antibodysurface. Frequently, however, less exposed locations are preferred forengineered cysteine residues in ecmAbs used in antibody conjugates suchas ADCs. Reducing agents can react with these less exposed engineeredcysteine residues and with disulfide bonds at comparable rates. As such,it is often necessary to find selective activation conditions whenreactions (a) and (b) occur at comparable rates.

In cases where reactions (a) and (b) occur at comparable rates—or wherereaction (b) is faster than reaction (a}-selective activation of theengineered cysteine requires ensuring that when an inter-chain disulfideis attacked by the reducing agent, the reaction (c) to re-form thedisulfide is faster than the second reduction step (d), which results inthe undesired cleavage of an inter-chain disulfide. In general, it isnot possible to impact the rate of reaction (c), because it is aunimolecular reaction. The thiol and mixed disulfide shown in 6 are partof the same antibody. The rate of reaction (d), however, is dependent onthe concentration of reductant, so reaction (d) can be made to be slowerthan reaction (c) by using a sufficiently low concentration of reducingagent. Of course, reactions (a) and (b) are also dependent on theconcentration of reducing agent, so under these conditions activation isalso slow. Hence, selective activation of engineered cysteines isgenerally favored by the maintenance of very mild reduction conditions(low concentration of reducing agent, and relatively mild reducingagent) over a long period of time.

Reduction reaction mixtures can include any suitable amount of protein.Typically, the concentration of protein (whether antibody ornon-antibody protein) in the reduction reaction mixture ranges fromabout 0.01 mg/mL to about 150 mg/mL, more typically from about 1 mg/mlto about 50 mg/ml. The reduction reaction mixture can contain, forexample, about 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,7.0, 8.0, 9.0, 10.0, 12.5, 15, 17.5, 20, 22.5, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or about150 mg of protein (whether antibody or non-antibody protein) per mL ofreduction reaction mixture. The concentration of protein (whetherantibody or non-antibody protein) can be higher or lower, depending onthe particular reaction conditions employed. One of skill in the artwill be able to convert a mass-based concentration (e.g., mg/mL) to amolar concentration (i.e., moles/L).

Any suitable amount of reducing agent can be used in the methods ofinvention. In general, the concentration of the reducing agent in thereduction reaction mixture is high enough that the reaction proceeds,but low enough that inter-chain disulfide reduction is negligibly slow.The reduction reaction mixture is typically formed using an initialamount of the reducing agent at an initial concentration. This initialconcentration is substantially maintained by supplementing the reductionreaction mixture with additional amounts of the reducing agent in acontinuous or step-wise fashion through the duration of the reductionreaction. In certain embodiments, the reduction reaction mixture issupplemented with additional amounts of the reducing agent in acontinuous fashion throughout the reduction reaction. The optimalconcentration of reducing agent can be experimentally determined usingthe teachings described herein, and will be different for differentthiols because of their different reduction strengths. It was determinedthat an optimal concentration of cysteine using the conditions describedin the examples, is about 0.5 mM to about 1.5 mM. Depending on thestrength of the reducing agent and the concentration of ecmAb, thatconcentration may be increased or decreased. In some embodiments, theconcentration of reducing agent will be maintained at a concentrationgreater than the concentration of total antibody. The optimalconcentration ratio of reducing agent to total antibody will also bedependent on the strength of the reducing agent. The concentration ofreducing agent in the reduction reaction mixture will, in some aspects,be about 5 times to about 25 times, 5 times to about 20 times, 5 timesto about 15 times, or 5 times to about 10 times higher than theconcentration of total antibody in the reduction reaction mixture. Forexample, in some embodiments, the concentration ratio of reducing agentto total antibody in the reduction reaction mixture will be about 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1,18:1, 19:1, or 20:1. In some embodiments, including some embodimentswhen the reducing agent is cysteine, the concentration ratio of reducingagent to total antibody in the reduction reaction mixture will be fromabout 5:1 to about 12:1, from about 7:1 to about 10:1, preferably about8:1. In some such aspects, the engineered cysteine residue will be atposition 239 of the heavy chain (numbering according to the EU index).Other concentration ratios can be used in the methods of the invention,depending on factors such as the particular reducing agent being used orthe location of the engineered cysteine residue in the antibody.

The reduction reaction to uncap the cysteine residues (engineered ornative cysteine residues) is conducted such that it minimizes reductionof the interchain disulfide bonds and the subsequent generation of freethiols. Substantially all of the inter-chain disulfide bonds present inthe protein having the capped cysteine residues are retained in theuncapped protein preparation formed using the present methods. Ingeneral, at least about 80% of the inter-chain disulfide bonds areretained in the uncapped protein preparation. In certain embodiments, atleast about 85% of the inter-chain disulfide bonds are retained in theuncapped protein preparation. In certain embodiments, at least about 90%or about 95% of the inter-chain disulfide bonds are retained in theuncapped protein preparation. In certain embodiments, all of theinter-chain disulfide bonds are retained in the uncapped proteinpreparation. Or, in other words, in exemplary embodiments, no more thanabout 20%, no more than about 15%, no more than about 10%, or no morethan about 5% of the inter-chain disulfide bonds are converted duringthe reduction reaction to a pair of free thiols. As it applies toantibodies, substantially all of the inter-chain disulfide bonds presentin the antibody molecules having the capped engineered cysteine residues(e.g., antibody molecules prior to the reduction reaction) are retainedin the uncapped antibody preparation formed using the present methods.In general, at least about 80% of the inter-chain disulfide bonds areretained in the uncapped antibody preparation. In certain embodiments,at least about 85% of the inter-chain disulfide bonds are retained inthe uncapped antibody preparation. In certain embodiments, at leastabout 90% or about 95% of the inter-chain disulfide bonds are retainedin the uncapped antibody preparation. In certain embodiments, all of theinter-chain disulfide bonds are retained in the uncapped antibodypreparation. Or, in other words, in exemplary embodiments, no more thanabout 20%, no more than about 15%, no more than about 10%, or no morethan about 5% of the inter-chain disulfide bonds are converted duringthe reduction reaction to a pair of free thiols.

While the concentration of the reducing agent is maintained in thereduction reaction mixture, reduction byproducts are removed from thereduction reaction mixture. Reducing agent addition and byproductremoval can be conducted in a continuous or step-wise fashion. Incertain embodiments, reducing agent addition and byproduct removal areconducted in a continuous fashion. Reducing agent can be added andbyproducts can be removed by techniques including, but not limited to,tangential flow filtration (e.g., diafiltration) size exclusionchromatography, solid phase immobilization, and dialysis. In adiafiltration process, an aqueous reducing agent solution is typicallyadded to the reduction reaction mixture at a given flow rate while aportion of the mixture is removed at about the same flow rate. Asemi-permeable membrane can be used to retain the protein (e.g.,antibody) in the reduction reaction mixture. Alternatively, the protein(e.g., antibody) can be immobilized on a solid support (such as ProteinA agarose beads) and the aqueous reducing agent solution can be passedacross the immobilized protein (e.g., antibody). A reactive resin thatreacts with the cap byproduct can also be used. For example, capbyproduct can be sequestered using resins that have reactive moietiessuch as disulfides on the interior of pores small enough to exclude theprotein (e.g., antibody).

Accordingly, some embodiments of the invention provide methods asdescribed above wherein removing the cap byproduct from the reductionreaction mixture comprises dialyzing or diafiltering the reductionreaction mixture during the reduction reaction. In some embodiments,removing the cap byproduct from the reduction reaction mixture comprisesdiafiltering the reduction reaction mixture during the reductionreaction.

Often, the methods of the invention are conducted so as to maximize theuncapping of cysteine residues. Any one protein molecule, however, canhave capped cysteine residues as well as uncapped residues. Often, anuncapped antibody preparation will contain two or more of: an antibodymolecule having at least two uncapped engineered cysteine residues andno capped engineered cysteine residues; an antibody molecule having atleast two capped engineered cysteine residues and no uncapped engineeredcysteine residues; and an antibody molecule having at least one cappedengineered cysteine residue and at least one uncapped engineeredcysteine residue. Advantageously, the methods of the invention providehigh levels of selectively-uncapped engineered cysteine residues. Forexample, the methods can be conducted such that at least 40% of theengineered cysteine residues are uncapped. In some aspects, the methodscan be conducted such that at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, or at least about 90% of the engineered cysteineresidues are uncapped. In certain embodiments, at least 60% of theengineered cysteine residues are uncapped. Higher or lower levels ofuncapping can also occur, depending in part on the particular antibodyas well as the location of the engineered cysteine residues within theantibody. Although, the methods of the invention are generally conductedso as to maximize the uncapping of cysteine residues, there may beinstances when maximizing uncapping of cysteine residues is not desired,for example, with an antibody having two engineered cysteine residueswhen it is desired to only conjugate one of the cysteine residues to afunctional agent. In some such embodiments, the present methods can beused to tightly control activation of engineered cysteine residues. Anadvantageous aspect of the method of the invention is that intermediatelevels of uncapping are readily attainable simply by stopping theactivation at the desired level and conjugating. Such intermediateconjugates are difficult to obtain when using the method of completereduction followed by re-oxidation.

Reaction mixtures can contain additional reagents or components. Asnon-limiting examples, the reaction mixtures can contain buffers (e.g.,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), co-solvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, isopropanol, glycerol,tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g.,NaCl, KCl, CaCh, and salts of Mn2+ and Mg2+), denaturants (e.g., ureaand guandinium hydrochloride), detergents (e.g., sodium dodecylsulfateand Triton-X 100), and chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid(BAPTA)). Buffers, co-solvents, salts, denaturants, detergents, andchelators can be used at any suitable concentration, which can bereadily determined by one of skill in the art. In general, buffers,co-solvents, salts, denaturants, detergents, and chelators if present,are included in reaction mixtures at concentrations ranging from about 1μM to about 1 M. For example, a buffer, a co-solvent, a salt, adenaturant, a detergent, or a chelator can be included in a reactionmixture at a concentration of about 1 μM, or about 10 μM, or about 100μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, orabout 100 mM, or about 250 mM, or about 500 mM, or about 1 M.Cosolvents, in particular, can be included in the reaction mixtures inamounts ranging from, for example, about 1% v/v to about 75% v/v, orhigher. A cosolvent can be included in the reaction mixture, forexample, in an amount of about 5, 10, 20, 30, 40, or 50% v/v.

Reactions are conducted under conditions sufficient to form uncappedcysteine residues. The reactions can be conducted at any suitabletemperature. In general, the reactions are conducted at a temperature offrom about 4° C. to about 40° C. The reactions can be conducted, forexample, at about 25° C. or about 37° C. The reactions can be conductedat any suitable pH. In general, the reactions are conducted at a pH offrom about 6.5 to about 10. In certain instances, the pH is from about7.0 to about 8.5. The reactions can be conducted for any suitable lengthof time. The length of time selected will be dependent on the strengthof the reducing agent. Because mild reduction conditions (lowconcentration of reducing agent, and relatively mild reducing agent) areused in the present methods, the reduction reaction will extend longerthan typically expected for reduction of inter-chain disulfide bonds. Insome preferred aspects, the reduction reaction will proceed until atleast about 60%, at least about 70% or at least about 80% or at leastabout 85% of the capped engineered cysteine residues are uncapped. Insome aspects, the reduction reaction will be incubated under suitableconditions for at least one hour, at least two hours, at least threehours, at least four hours, at least five hours, or at least about 6hours. Reactions can be conducted under an inert atmosphere, such as anitrogen atmosphere or argon atmosphere. Other reaction conditions canbe employed in the methods of the invention, depending on the identityof a particular antibody, or reducing agent.

In some embodiments, the pH of the reaction mixture ranges from about6.5 to about 8.5. In some embodiments, the methods of the inventioninclude maintaining the reaction mixture at a temperature ranging fromabout 4° C. to about 37° C. In some embodiments, the reaction mixture ismaintained at a desired temperature for a period of time ranging fromabout 1 hour to about 8 hours.

A number of known purification techniques can be employed at variouspoints during the methods of the invention. Such techniques can be usedto remove excess reducing agents, to exchange buffers or othercomponents into and out of reaction mixtures, and to concentrate ordilute antibody compositions as necessary. Purification techniquesuseful in the methods of the invention include, but are not limited to,tangential flow filtration (TFF), gel filtration, immunoprecipitation,affinity chromatography, and the like. Preferably, purification time isminimized to prevent unwanted oxidation or re-capping of uncappedengineered cysteine residues.

Antibody Conjugates

Uncapped engineered cysteine residues on an antibody serve as usefulhandles for installation of a variety of functional groups, includingimaging agents (such as chromophores and fluorophores), diagnosticagents (such as MRI contrast reagents and radioisotopes), stabilityagents (such as polyetheylene glycol polymers) and therapeutic agents.Antibodies having uncapped cysteine residues can be conjugated tofunctional agents to form antibody-functional agent-conjugates. Thefunctional agent (e.g., drug, detection agent, stability agent) isconjugated (covalent attachment) to the antibody at the site of anengineered cysteine residue. A functional agent can be attachedindirectly via a linker or directly via a thiol-reactive group on thefunctional agent.

Antibodies having uncapped cysteine residues can be conjugated to drugsto form antibody drug conjugates (ADCs). Typically, the ADC contains alinker between the drug and the antibody. The linker can be a cleavableor a non-cleavable linker. A cleavable linker is typically susceptibleto cleavage under intracellular conditions such that cleavage of thelinker releases the drug from the antibody at the target site. Suitablecleavable linkers include, for example, enzyme cleavable linkersincluding peptidyl containing linkers cleavable by an intracellularprotease, such as lysosomal protease or an endosomal protease or sugarlinkers for example, glucuronide containing linkers cleavable by aglucuronidase. Peptidyl linkers can include, for example, a dipeptide,such as valine-citrulline (val-cit) phenylalanine-lysine (phe-lys) orvaline-alanine (val-ala). Other suitable cleavable linkers include, forexample, pH-sensitive linkers (e.g., linkers hydrolyzable at a pH ofless than 5.5, such as a hydrazone linker) and linkers cleavable underreducing conditions (e.g., disulfide linkers). Non-cleavable linkerstypically release drugs by proteolytic degradation of the antibody.

Prior to attachment to the antibody, the linker will have a groupreactive with the uncapped engineered cysteine residues and attachmentwill be via the reactive group. Thiol-specific reactive groups arepreferred and include, for example, maleimides; haloacetamides (e.g.,iodo, bromo or chloro); haloesters (e.g., iodo, bromo or chloro);halomethyl ketones (e.g., iodo, bromo or chloro); benzylic halides(e.g., iodide, bromide or chloride); vinyl sulfones;(pyridyl)disulfides; disulfide dioxide derivatives; mercury derivativessuch as 3,6-bis-(mercurimethyl)dioxane with counter ions of acetate,chloride or nitrate; and polymethylene bismethane thiosulfonates. Thelinker can include, for example, a maleimide that attaches to theantibody via a thio-succinimide linkage.

The drug can be any cytotoxic, cytostatic or immunosuppressive drug. Inembodiments wherein a linker links the antibody and the drug, the drughas a functional group that can form a bond with the linker. Forexample, the drug can have an amine, a carboxylic acid, a thiol, ahydroxyl group, or a ketone that can form a bond with the linker. Inaspects wherein the drug is directly attached to the linker, the drugwill, prior to attachment to the antibody, have a group reactive withthe uncapped engineered cysteines.

Useful classes of drugs include, for example, antitubulin agents, DNAminor groove binders, DNA replication inhibitors, alkylating agents,antibiotics, antifolates, antimetabolites, chemotherapy sensitizers,topoisomerase inhibitors, vinca alkaloids, or the like. Particularlyexamples of useful classes of cytotoxic agents include, for example, DNAminor groove binders, DNA alkylating agents, and tubulin inhibitors.Exemplary cytotoxic agents include, for example, auristatins,camptothecins, duocarmycins, etoposides, maytansines and maytansinoids(e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepinecontaining drugs (e.g., pyrrolo[1,4]-benzodiazepines (PBDs),indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vincaalkaloids. Select benzodiazepine containing drugs are described in WO2010/091150, WO 2012/112708, WO 2007/085930, and WO 2011/023883.

In some typical embodiments, suitable cytotoxic agents include, forexample, DNA minor groove binders (e.g., enediynes and lexitropsins, aCBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins (see U.S.Publication No. 20060024317), taxanes (e.g., paclitaxel and docetaxel),puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

The drug can be an anti-tubulin agent. Examples of anti-tubulin agentsinclude, but are not limited to, taxanes (e.g., Taxol® (paclitaxel),Taxotere® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulinagents include, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, maytansinoids, combretastatins,discodermolide, auristatins, and eleutherobin.

The drug can be a maytansine or a maytansinoid, another group ofanti-tubulin agents. (ImmunoGen, Inc.; see also Chari et al., 1992,Cancer Res. 52:127-131 and U.S. Pat. No. 8,163,888).

The drug can be an auristatin. Auristatins include, but are not limitedto, AE, AFP, AEB, AEVB, MMAF, and MMAE. The synthesis and structure ofauristatins are described in U.S. Patent Application Publication Nos.2003-0083263 and 2009-0111756; International Patent Publication No. WO04/010957; International Patent Publication No. WO 02/088172; U.S. Pat.Nos. 6,884,869; 7,659,241; 7,498,298; 8,343,928; and 8,609,105; each ofwhich is incorporated by reference in its entirety and for all purposes.

In some embodiments, the drug moiety is selected from the groupconsisting of an anti-tubulin agent, a DNA binding agent, and a DNAalkylating agent. In some embodiments, the drug is selected from thegroup consisting of an auristatin, a pyrrolobenzodiazepine, aduocarmycin, a maytansinoid, a taxane, a calicheamicin, and ananthracycline.

A drug-linker can be used to form an ADC in a single step. In otherembodiments, a bifunctional linker compound can be used to form an ADCin a two-step or multi-step process. In one example, the uncappedengineered cysteine residue is reacted with the reactive moiety of alinker in a first step, and a functional group on the linker is reactedwith a drug to form the ADC in a subsequent step.

Generally, a functional group on the linker is selected for specificreaction with a suitable reactive group in the drug moiety. As anon-limiting example, an azide-based moiety can be used for specificreaction with a reactive alkyne group in the drug moiety. The drug iscovalently bound to the linker via 1,3-dipolar cycloaddition of theazide and alkyne. Other useful functional groups include, for example,ketones and aldehydes (suitable for reaction with hydrazides andalkoxyamines); phosphines (suitable for reaction with azides);isocyanates and isothiocyanates (suitable for reaction with amines andalcohols); and activated esters such as N-hydroxysuccinimidyl esters(suitable for reaction with amines and alcohols). These and otherlinking strategies, as described, for example, in BioconjugateTechniques, 2^(nd) Ed. (Elsevier), are well known to those of skill inthe art. One of skill in the art will appreciate that when acomplementary pair of reactive functional groups is chosen for selectivereaction of the drug moiety to the linker, each member of the pair canbe employed on either the linker or the drug.

Accordingly, some embodiments of the invention provide methods forpreparing an uncapped antibody preparation as described above, furtherincluding combining uncapped antibody (e.g., combining the uncappedantibody preparation) with a drug-linker compound under conditionssufficient to form an antibody-drug conjugate (ADC).

In some embodiments, the methods include combining uncapped antibodywith a bifunctional linker compound, under conditions sufficient to forman antibody-linker conjugate. In such embodiments, the methods of theinvention can further include combining the antibody-linker conjugatewith a drug moiety under conditions sufficient to covalently link thedrug moiety to the antibody via the linker.

In some embodiments, the ADC is of the following formula:

wherein

Ab is anantibody,

LU is a linker,

D is a drug;

and the subscript p is a value from 1 to 8.

In the formula above, the linker, LU, is conjugated to the antibody viathe uncapped engineered cysteines. The value of the subscript p isdependent on the number of uncapped engineered cysteines available forconjugation. For example, for an antibody having two uncapped engineeredcysteines, (e.g., one site on each heavy chain or one site on each lightchain), the value of p can be two. Similarly, for an antibody havingfour uncapped engineered cysteines (e.g., two sites on each heavy chain,or two sites on each light chain, or one site on each heavy chain andone site on each light chain), the value of p can be four. In somepreferred embodiments, p is a value from 1 to 4.

Drug Loading

The average number of drug-linker molecules per antibody (or averagedrug load) is an important characteristic of an ADC composition, as itis a primary determinant of the amount of drug that can be delivered toa target cell. The average drug load includes drugs conjugated toengineered cysteine residues, as well as drugs conjugated to sites otherthan the intended engineered cysteine residues and the amount ofunconjugated antibodies in the composition. When an average drug loadingof about two drugs per antibody is targeted, antibodies having twoengineered engineered cysteine residues (e.g., one site on each heavychain or one site on each light chain) can be used to prepare the ADCcomposition. When an average drug loading of about four drugs perantibody is targeted, antibodies having four engineered cysteineresidues (e.g., two sites on each heavy chain, or two sites on eachlight chain, or one site on the heavy chain and one site on the lightchain) can be used to prepare the ADC composition. One of skill in theart will appreciate that other levels of drug loading can betherapeutically useful depending on the particular antibody or theparticular drug (including, for example, drug loading levels less than 2as well as drug loading levels greater than 4). Sites for drugconjugation can be introduced in an antibody by placing engineeredcysteines at more than one site or more than two sites in the heavychain, or by placing an engineered cysteine in the light chain, or both.Importantly, the level of engineered cysteine residue uncappingdescribed above allows for preparation of ADC compositions with usefuldrug loading.

Typically, ADC compositions prepared with antibodies having twoengineered cysteine residues have an average drug-loading of from about1.5 to 2.5 drugs per antibody. The average number of drug moieties perantibody can be, for example, about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, or 2.5. In some embodiments, the average drug-loading forADC compositions prepared with antibodies having two engineered cysteineresidues is from about 1.5 to about 2.2 drug moieties per antibody, orfrom about 1.8 to about 2 drug moieties per antibody. Typically, ADCcompositions prepared with antibodies having four engineered cysteineresidues have an average drug-loading of from about 3.4 to 4.5 drugmoieties per antibody. The average number of drug moieties per antibodycan be, for example, about 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. Insome embodiments, the average drug-loading for ADC compositions preparedwith antibodies having four engineered cysteine residues is from about3.6 to about 4.2 drug moieties per antibody, or from about 3.8 to about4 drug moieties per antibody.

The methods of the invention are typically conducted so as toselectively modify uncapped engineered cysteine residues, but varyingdegrees of non-engineered (i.e., native) cysteine residue modificationare commonly observed. Reaction conditions can be controlled to limitthe modification of non-engineered cysteine residues as necessary. Incertain instances, the methods can be conducted to eliminate or minimizemodification of non-engineered cysteine residues. For example, themethods can be conducted such that no more than about 20%, no more thanabout 15%, no more than about I 0%, or no more than about 5% of theantibody molecules in the ADC composition have a drug moiety covalentlylinked to a non-engineered cysteine residue. The methods can also beused to prepare ADC compositions wherein the number of antibodymolecules with modified non-engineered cysteine residues amount to nomore than about 5%, 10%, 15%, 20%, 25%, or 30% of the total antibodymolecules.

Various analytical methods can be used to determine the yields andisomeric mixtures of the conjugates. Following conjugation of the drugto the antibody, the conjugated drug-antibody species can be separated.In some embodiments, the conjugated antibody species can be separatedbased on the characteristics of the antibody, the drug and/or theconjugate. Other techniques useful for analysis of ADC compositionsinclude, but are not limited to, reversed-phase chromatography,capillary electrophoresis, and mass spectrometry. ADC compositions canbe analyzed, for example, by LC/MS coupled with proteolytic digestion todetermine the location of a drug moiety in an ADC.

Antibodies

A number of suitable antibodies can be used in the methods of theinvention. Antibodies used in the methods of the invention are usefulfor a number of applications, including in vitro or in vivo diagnosis,in vivo imaging, and therapy for diseases and conditions associated withdistinctive antigens. Five human antibody classes (IgG, IgA, IgM, IgDand IgE), as well as various subclasses (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) within these classes, are recognized on the basis ofstructural differences, such as the number of immunoglobulin units in asingle antibody molecule, the disulfide bridge structure of theindividual units, and differences in chain length and sequence. Theclass and subclass of an antibody is referred to as the antibody'sisotype.

The antibody can be an intact antibody or an antigen-binding antibodyfragment, provided that the antibody fragment contains at least oneinter-chain disulfide bond.

Typically, the antibodies are human, rodent (e.g., mouse and rat),donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken. Theantibody can be, for example, a murine, a chimeric, humanized, or fullyhuman antibody produced by techniques well-known to one of skill in theart. Recombinant antibodies, such as chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, which can bemade using standard recombinant DNA techniques, are useful antibodies. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal and human immunoglobulin constantregions. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Bosset al., U.S. Pat. No. 4,816,397, which are incorporated herein byreference in their entirety.) Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. (See, e.g., Queen, U.S.Pat. No. 5,585,089, which is incorporated herein by reference in itsentirety.) Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries, from human B cells, or from animals transgenicfor one or more human immunoglobulin, as described for example in U.S.Pat. Nos. 5,939,598 and 6,111,166.

The antibodies may be monospecific, bispecific, trispecific, or ofgreater multispecificity.

In certain instances, the constant domains have effector function. Theterm antibody effector function, as used herein refers to a functioncontributed by an Fe domain(s) of an lg. Such function can be effectedby, for example, binding of an Fe effector domain(s) to an Fe receptoron an immune cell with phagocytic or lytic activity or by binding of anFe effector domain(s) to components of the complement system. Theeffector function can be, for example, “antibody-dependent cellularcytotoxicity” or ADCC, “antibody-dependent cellular phagocytosis” orADCP, “complement-dependent cytotoxicity” or CDC. In certain instances,the constant domain lack one or more effector functions. Conjugation ofa drug-linker compound to an engineered cysteine residue located in aneffector function binding domain can modulate the effector function.

The antibodies may be directed against any antigen of interest, such asof medical and/or therapeutic interest. For example, the antigen can beone associated with pathogens (such as but not limited to viruses,bacteria, fungi, and protozoa), parasites, tumor cells, or particularmedical conditions. In the case of a tumor-associated antigen (TAA), thecancer may be of the immune system, lung, colon, rectum, breast, ovary,prostate gland, head, neck, bone, or any other anatomical location.Antigens of interest include, but are not limited to, CD30, CD40, LewisY, CD70, CD2, CD20, CD22, CD33, CD38, CD40, CD52, HER2, EGFR, VEGF, CEA,HLA-DR, HLA-Dr10, CA125, CA15-3, CA19-9, L6, Lewis X, alpha fetoprotein,CA 242, placental alkaline phosphatase, prostate specific antigen,prostatic acid phosphatase, epidermal growth factor, MAGE-I, MAGE-2,MAGE-3, MAGE-4, anti-transferrin receptor, p97, MUCI-KLH, gp100, MARTI,IL-2 receptor, human chorionic gonadotropin, mucin, P21, MPG, and Neuoncogene product.

Some specific useful antibodies include, but are not limited to,antibodies against the CD33 antigen (e.g., a humanized 2H12 antibody asdescribed in International Application Number WO 2013/173496),antibodies against the CD70 antigen, (e.g., a humanized 1F6 antibody asdescribed in International Application Number WO2006/113909), antibodiesagainst the CD30 antigen (e.g., a humanized ACI0 antibody as describedin International Application Number WO2008/025020), antibodies againstthe CD19 antigen (e.g., a humanized BU12 antibody as described inInternational Application Number WO 2009/052431), antibodies againstLIV-I, NTBA, or alpha V Beta 6. Many other internalizing antibodies thatbind to tumor specific antigens can be used, and have been reviewed(see, e.g., Franke et al. (2000), Cancer Biother Radiopharm. 15:459-76;Murray (2000), Semin Oncol. 27:64-70; Breitling et al., RecombinantAntibodies, John Wiley, and Sons, New York, 1998). The disclosures ofthese references and International Applications are incorporated byreference herein and for all purposes.

In some embodiments, the invention provides methods for preparing anantibody having an uncapped engineered cysteine residue as describedabove, wherein the antibody comprises at least three inter-chaindisulfide bonds. In some embodiments, the antibody comprises at leastfour inter-chain disulfide bonds. In some embodiments, the antibodycomprises 1, 2, 3, 4, or 5 inter-chain disulfide bonds. In someembodiments, the engineered cysteine residue is present in the heavyconstant region or the light constant region of the antibody.

Engineered Cysteine Sites

The site of the engineered cysteine can have an impact on the propertiesof the ADC.

For instance, engineered cysteines entirely buried in the structure ofthe protein can be difficult to conjugate because of poor access to thesolvent, while engineered cysteines on the exterior surface of theantibody may result in ADCs that have impaired stability because ofprolonged exposure to materials in plasma. Also, ADCs prepared fromecmAbs with highly surface exposed engineered cysteines may be sensitiveto the hydrophobicity of the drug, while engineered cysteines in moreprotected locations may be less sensitive to the properties of the drug,because access to other materials in solution is restricted. Thelocation of an engineered cysteine residue can also be used to modulateeffector function as desired for a particular ADC. For example,conjugation of a drug-linker to an engineered cysteine residue in aneffector function binding domain can be used to block binding toeffector function-mediating receptors.

In some embodiments, the engineered cysteine is located in the heavychain constant region, the heavy chain variable region, the light chainvariable region, the light chain constant region, or combinationsthereof. Preferred engineered cysteine residues are residues that arelocated at sites that are conjugatable and result in stable linkages. Byconjugatable it is meant that the engineered cysteine residue is capableof being conjugated to a functional agent (e.g., imaging agents,diagnostic agents, stability agents or therapeutic agents) without firstdenaturing the antibody. Methods for selecting a site for introducing acysteine residue that can be subsequently conjugated to a functionalagent are known in the art (e.g., See, for example, Junutula et al.,2008, Nature Biotechnology, 26(8), 925-932)

In some aspects, the engineered cysteine residue is one that has afractional solvent accessibility of 10% or above, 20% or above, 30% orabove, 40% or above, or 50% or above. In some aspects, the cysteineresidue is one that has a fractional solvent accessibility of from about10% to about 95%, from about 10% to about 85%, from about 10% to about75%, from about 10% to about 60%, from about 20% to about 95%, fromabout 20% to about 85%, from about 20% to about 75%, from about 20% toabout 60%, or from about 40% to about 95%, from about 40% to about 85%,from about 40% to about 75%, from about 40% to about 60%. Methods fordetermining the fractional solvent accessibility of a residue at aparticular site are known in the art and can be determined, for example,using the online server get area that uses the methodology described inFraczkiewicz and Braun, 1998, J. Comp. Chem., 19, 319-333 (seehttp://curie.utmb.edu/getarea.html). Exemplary residues include those atsites 15, 114, 121, 127, 168, 205, on the light chain (numberingaccording to Kabat) or sites 112, 114, or 116 on the heavy chain(numbering according to Kabat numbering). Exemplary residues includesthose in the Fe region of an IgG1 antibody such as those at sites 239,326, 327, or 269 in the Fe region (numbering according to the EU index).The fraction solvent accessibility of residues at site 239, 326, and 327is about 50%, about 94%, and about 23%, respectively.

One of skill in the art will recognize that the conditions required forselective activation of the engineered cysteine will be dependent on thesite of the engineered cysteine in the antibody. The chemical formulasin Scheme 2 provide a framework for selecting reduction conditions thatenable selective activation of engineered cysteines, wherever they occurin the protein sequence. In some embodiments, an antibody has from 1 to8 or from 2 to 8 or from 2 to 4 engineered cysteine residues.

Non-Antibody Proteins

It will be appreciated by those skilled in the art that although theprocess described herein is exemplified with respect to antibodies, itmay be successfully employed for any protein with unpaired cysteines(cysteines that do not generally form inter-chain or intra-chain bondswithin the protein), engineered or native, that are capped with thiolsduring expression or production. Proteins for which this process isparticularly helpful are proteins, that, in addition to comprisingunpaired cysteines, contain native cysteines that form inter-chaindisulfide bonds, particularly bonds that can be cleaved withoutimmediately resulting in unfolding of the protein. When referring to anon-antibody protein, the term inter-chain disulfide bond refers to acovalent bond between two cysteine residues on adjacent polypeptidechains. Candidate non-antibody proteins include those which containsolvent exposed disulfide bonds whose stability in native foldedconformation is comparable to those of the capped thiols. An engineeredcysteine protein, as used herein, is one in which selected amino acidsin the protein have been mutated to cysteine. Exemplary proteins alsoinclude Fe-fusion proteins, e.g., protein containing a Fe region of anantibody covalently linked to a protein that provides specificity for adesired target.

IV. Examples Example 1: An Exemplary Method for Performing SelectiveReduction

A reaction vessel is connected to a tangential flow filtration (TFF)apparatus, and an ultrafiltration membrane is installed (e.g., 88 cm²,Millipore Pelican 3, regenerated cellulose). (Many different membranetypes with appropriate sizes can be used in this process The efflux rateand the sieving factor required for the addition rate calculation forthe reducing agent should be determined at the permeate flux and withthe membrane type and surface area that will be used in the process).The membrane is flushed and equilibrated according to the manufacturer'sinstructions. A diafiltration buffer reservoir is set up containingbuffer (e.g., 50 mM Tris/5 mM EDTA, pH 8.0). It is connected via tubingto the reaction vessel, and flow through the tubing is controlled by aperistaltic pump (e.g., diafiltration buffer pump). A mAb containingcapped engineered cysteines is placed in the reaction vessel. Thereaction is performed at room temperature with the reaction mixturecontinuously pumped past the ultrafiltration membrane, with theretentate line constricted to maintain a trans-membrane pressure of −20psi.

A reservoir containing a solution of the reducing agent is connected viatubing to the diafiltration buffer line, or some other location in theflow path or reaction vessel. A flow rate is calculated at which thestock solution containing the reducing agent should be added to thereaction mixture to maintain the initial (desired) concentration ofreducing agent in the reaction mixture. (The optimal concentration ofreducing agent in the reaction mixture is experimentally determinedusing the teachings described herein, and will be different fordifferent thiols because of their different reduction strengths asdemonstrated in FIG. 1 .) The concentration of the reducing agent shouldremain largely constant over the entire reaction time. Diafiltrationbuffer is also pumped into the reaction vessel at a rate that iscontrolled so that the total reaction volume remains constant (as inconstant-volume-diafiltration), i.e. the rate of introduction ofreducing agent and diafiltration buffer into the reactor matches therate of volume loss through the permeate.

Example 2. Uncapping Engineered Cysteine Residues Using Cysteine,N-Acetyl Cysteine and Cysteamine as Reducing Agents without Removal ofCap Byproduct

A S239C engineered cysteine antibody (IgG I antibody with an engineeredcysteine residue at site 239, numbering according to the EU index), 20mg (135 nmol at 15 mg/mL) was treated with 33 μL 100 mM (3.3 μmol)cysteine, N-acetyl cysteine, or cysteamine for 8 hours at pH 8.0 androom temperature. Samples were removed at I hour intervals, purified,conjugated with excess SGD-1269, and stored frozen. Purification andconjugation stopped the reduction reaction, preserving thiols that hadbeen generated by the reduction procedure. The conjugated samples wereanalyzed by reversed phase HPLC under denaturing conditions (“rPLRP”),from which the extent of engineered cysteine uncapping could be deduced(conversion of HO to HI). The extent of inter-chain disulfide cleavagecould also be determined from the amount of heavy chain with >I mcMMAFconjugated (mcMMAF is the maleimido caproic acid linker attached to thedrug monomethylauristatin F). No heavy chain was observed with >2 mcMMAFmolecules conjugated. Results from the static solution reductionexperiments are provided in FIG. 1 . The results show that each of thethree thiols can react with the ecmAb to uncap the engineered cysteine,but that the three behave quite differently. NAC behaves in a mannerthat is most directly described by the first reaction in Scheme I: Thereaction proceeds until a sufficient concentration of disulfide:!:. hasaccumulated, then stops because the forward and reverse reactions areoccurring at the same rate. Cysteine behaves as a more powerfulreductant than NAC, but instead of stopping at partially activatedecmAb, the reaction reverses, regenerating the ecmAb. This reversalindicates that, with cysteine, an additional reaction is involved,namely autoxidation of the reducing agent.

2R—SH+02R—S—S—R  (iii)

Thus, cysteine, produced by autoxidation of cysteine, also re-capsactivated engineered cysteines, reversing the initial reduction.Examination of Figure I shows that this same phenomenon, initialreduction followed by re-capping, also occurs with cysteamine, but thatcysteamine is a weaker reducing agent, so that the initial extent ofreduction is not as high as with cysteine or N-acetylcysteine.

Example 3. Selective Activation of Engineered Cysteine Residues inecmAbs

A reaction vessel was connected to a tangential flow filtration (TFF)apparatus, and ultrafiltration membranes were installed (88 cm²,Millipore Pelican 3, regenerated cellulose). The membrane was flushedand equilibrated according to the manufacturer's instructions. Adiafiltration buffer reservoir was set up containing 50 mM Tris/5 mMEDTA, pH 8.0. It was connected via tubing to the reaction vessel, andflow through the tubing was controlled by a peristaltic pump(diafiltration buffer pump). The engineered cysteine mAb containingcapped engineered cysteines (h2Hl2 S239C ecMab, hlF6 239 ecMab, h2Hl2K326C ecMab or h2Hl2 A327C ecMab, numbering according to EU index) wasplaced in the reaction vessel at a concentration of approximately 15mg/mL and the pH was adjusted to 8.0. The reaction was performed at roomtemperature with the reaction mixture continuously pumped past theultrafiltration membrane, with the retentate line constricted tomaintain a trans-membrane pressure of −20 psi.

A reservoir containing cysteine solution was connected via tubing to thediafiltration buffer line. A flow rate was calculated at which thecysteine stock solution should be added to the reaction mixture tomaintain the initial (desired) concentration of cysteine in the reactionmixture. The concentration of the cysteine stock fed into the reactionmixture and the rate of addition were calculated as described below sothat the concentration of cysteine remained largely constant over theentire reaction time. In the examples illustrated in FIGS. 2-5 , thereaction concentration of cysteine was in the range of 0.5-0.9 mM (asdescribed in the figure descriptions); the stock concentration ofcysteine was either 100 mM, 10 mM, or 5 mM, and the flow rate ofcysteine pumped into the reactor was adjusted to maintain theexperimental cysteine concentration according to the formula below. Thesieving factor for the cysteine was measured at 0.8-0.9. Cysteineconcentration in the reaction mixture was determined periodicallythrough the experiment using the DTNB assay to ensure that the cysteinelevel remained at the desired concentration (not shown).

Diafiltration buffer was also pumped into the reaction vessel at a ratethat was controlled so that the total reaction volume remained constant(as in constant-volume-diafiltration), i.e. the rate of introduction ofcysteine and diafiltration buffer into the reactor matched the rate ofvolume loss through the permeate.

Samples were removed at the intervals indicated in the Figures, purifiedby elution over PD-10 columns, and conjugated with SGD-1269. Theconjugated samples were then analyzed by rPLRP. Analysis of the rPLRPchromatograms provided the fraction of heavy chain that remained capped(¾HO), the fraction that was selectively reduced at the engineeredcysteine (¾HI), or the fraction reduced at the engineered cysteine andadditionally at an inter-chain disulfide site (% H2; non-selectivereduction).

Cysteine Addition Rate Calculation

Cysteine was added to the reaction mixture at the same rate thatcysteine is lost through the permeate line. The initial rate of loss ofan analyte through the permeate line is calculated from Equation 2,which can be derived from the theoretical equation for clearance byconstant volume diafiltration, Equation 1. The rate at which cysteinestock solution is pumped into the reaction mixture to maintain theinitial cysteine concentration is then given by Equation 3.

C/C₀ =e(−NS).  Equation 1:

C is the concentration at any time, t; C₀ is the initial concentration;N is the number of diavolumes at time t, and Sis the sieving factor forthe analyte (determined empirically). The number of diavolumes, N, isr*t/Va, where is r is the permeate flow rate, and Va is the batchvolume. Making this substitution, taking the derivative, and evaluatingthe derivative at t=0, gives Equation 2.

dC/dt=−C₀*(r/Vd)*S,  Equation 2:

where dC/dt is the rate of loss of cysteine at t₀.

-   -   The rate at which cysteine solution must be added to achieve        given concentration in the reaction mixture is given by Equation        3.

R=−dC/dt*Va/[Cys],  Equation 3:

where [Cys] is the concentration of the cysteine stock solution.

Thus, substituting the expression for dC/dt in Equation 2, givesEquation 4, for the addition rate,

R═C₀ *r*S/[Cys]=(0.8 mM)*(720 mL/hr)*(0.8)/(100 mM)=4.61mL/hr.  Equation 4:

Results

Selective activation of engineered cysteines in the ecmAb was achievedby continuously diafiltering the ecmAb during the reaction, chargingcysteine into the diafiltration buffer throughout the process.Diafiltration continuously depleted the reaction byproduct. The cysteineaddition rate was calculated based on the theoretical rate of clearanceof cysteine by diafiltration, in order to provide a constantconcentration of cysteine in the reaction mixture. FIG. 2 shows −80%activation of available engineered cysteines (“¾H1”) after 4.5 hr withnegligible cleavage of inter-chain disulfide bonds (indicated by % H2).This experiment was repeated using 0.6 mM cysteine, for a longer time,providing a similar result (FIG. 3 ). At the higher concentration thereaction is faster and somewhat less specific. It will be appreciatedthat the reaction mixture is in a state of flux throughout the processwith cysteine solution entering the reaction mixture through thecysteine feed pump and exiting the reaction mixture through the TFFmembrane (permeate), so that the concentration can fluctuate somewhatover the course of the reaction, but nonetheless activation conditionscan generally be held close to the nominal concentrations. Cysteineconcentrations of 1.5-2 mM and higher resulted in increased inter-chaindisulfide cleavage. FIG. 4 demonstrates that engineered cysteineactivation can be accomplished selectively at a variety of sitesirrespective of the identity of the antibody. The activation time-coursethe K326 mutant indicates that activation is much faster at this sitethan at the S239 or A327 sites. This is likely due to the fact that theK326 site is more solvent accessible, but the method achieves thedesired selective activation, nonetheless. FIG. 5 illustrates that thereaction can be driven to very close to I 00% activation of theengineered cysteines with very good selectivity, by using a lowconcentration of reducing agent over a long period of time.

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

1.-43. (canceled)
 44. An uncapped engineered cysteine antibodypreparation prepared according to a method for the selective reductionof one or more capped engineered cysteine residues in intact antibodies,the method comprising: a) contacting a reducing agent with antibodymolecules, each of the antibody molecules having at least one cappedengineered cysteine residue and at least three heavy-light andheavy-heavy inter-chain disulfide bonds; b) reacting the reducing agentwith the antibody molecules under conditions sufficient to uncapengineered cysteine residues and form cap byproducts between thereducing agent and one or more cap moieties of the antibody molecules;and c) removing the cap byproducts during the reduction reaction;whereby an uncapped engineered cysteine antibody preparation is formedand at least about 80% of the heavy-light and heavy-heavy inter-chaindisulfide bonds present are retained in the uncapped engineered cysteineantibody preparation.
 45. The uncapped engineered cysteine antibodypreparation of claim 44, wherein the method comprises supplementing thereduction reaction with additional reducing agent while removing the capbyproduct.
 46. The uncapped engineered cysteine antibody preparationclaim 44 comprising two or more members selected from the groupconsisting of: an antibody molecule having at least two uncappedengineered cysteine residues and no capped engineered cysteine residues;an antibody molecule having at least two capped engineered cysteineresidues and no uncapped engineered cysteine residues; and an antibodymolecule having at least one capped engineered cysteine residue and atleast one uncapped engineered cysteine residue.
 47. The uncappedengineered cysteine antibody preparation of claim 44, wherein removingthe cap byproduct during the reduction reaction comprises dialysis ordiafiltration.
 48. The uncapped engineered cysteine antibody preparationof claim 44, wherein: each antibody molecule prior to the reductionreaction comprises four heavy-light and heavy-heavy inter-chaindisulfide bonds; each antibody molecule has at least two engineeredcysteine residues, or each antibody molecule has four engineeredcysteine residues.
 48. The uncapped engineered cysteine antibodypreparation of claim 44, wherein: the engineered cysteine residues arepresent in the heavy constant region of the antibody molecule. theengineered cysteine residues are present in the heavy chain or lightchain variable region of the antibody molecule.
 49. The uncappedengineered cysteine antibody preparation of claim 44, wherein: thereducing agent is selected from the group consisting of cysteine,cysteamine, (3-mercaptoethanol, 2-mercaptoethanesulfonic acid sodiumsalt, and mixtures thereof, or the reducing agent is cysteine.
 50. Theuncapped engineered cysteine antibody preparation of claim 49, wherein:the method comprises maintaining the reducing agent at a concentrationfrom about 5 times to about 15 times greater than the concentration ofthe antibody during the reduction reaction; or the method comprisesmaintaining the reducing agent at a concentration from about 5 times toabout 10 times greater than the concentration of the antibody during thereduction reaction.
 51. The uncapped engineered cysteine antibodypreparation of claim 49, wherein the concentration of cysteine ismaintained at a concentration of 0.5 mM to about 1.5 mM during thereduction reaction.
 52. The uncapped engineered cysteine antibodypreparation of claim 44, wherein the concentration of the cap byproductis maintained below the concentration at which re-capping preventsfurther activation of engineered cysteine residues.
 53. The uncappedengineered cysteine antibody preparation of claim 44, wherein theuncapped engineered cysteine antibody preparation is a monoclonalantibody preparation.
 54. The uncapped engineered cysteine antibodypreparation of claim 44: further comprising the step of removingresidual reducing agent from the uncapped engineered cysteine antibodypreparation; further comprising the step of purifying the uncappedengineered cysteine antibody preparation; or further comprisingcombining uncapped antibody with a drug-linker compound under conditionssufficient to form antibody-drug conjugate.
 55. The uncapped engineeredcysteine antibody preparation of claim 54, wherein: the average drugload of the antibody drug conjugate is about 3.6 to 4.2 drug moietiesper antibody; or the average drug load of the antibody drug conjugate isabout is about 2 drug moieties per antibody.
 56. The uncapped engineeredcysteine antibody preparation of claim 54, wherein the antibody drugconjugate is in solution.
 57. The uncapped engineered cysteine antibodypreparation of claim 44, wherein: at least about 85% of the heavy-lightand heavy-heavy inter-chain disulfide bonds present in the antibodymolecules prior to the reduction reaction are retained in the uncappedcysteine antibody preparation; or at least about 90% of the heavy-lightand heavy-heavy inter-chain disulfide bonds present in the antibodymolecules prior to the reduction reaction are retained in the uncappedcysteine antibody preparation.
 58. An activated engineered cysteineresidue prepared according to a method for the selective activation ofengineered cysteine residues of engineered cysteine antibodies, saidmethod comprising: (a) diafiltering a mixture of engineered cysteineantibodies and buffer, and (b) adding a reducing agent to said mixturein a concentration and at a rate to activate a portion of availableengineered cysteine residues while retaining at least 80% of allinter-chain disulfide bonds present in the engineered cysteineantibodies prior to (b); and (c) maintaining diafiltration of saidmixture during step (b); to selectively activate the engineered cysteineresidue.
 59. The activated engineered cysteine residue of claim 58,wherein less than 20% of said inter-chain disulfide bonds are convertedto a pair of free thiols.
 60. The activated engineered cysteine residueof claim 58, wherein said reducing agent is cysteine.
 61. The activatedengineered cysteine residue of claim 58, wherein at least 80% ofinter-chain disulfide bonds present in the engineered cysteineantibodies are retained prior to step (b).
 62. The activated engineeredcysteine residue of claim 59, wherein: less than 15% of said inter-chaindisulfide bonds are converted to a pair of free thiols; or less than 10%of said inter-chain disulfide bonds are converted to a pair of freethiols.